The present invention relates to an improved process for the enzymatic preparation of L-serine derivatives by the aldol condensation of glycine with an aldehyde. It further relates to an improved process for the preparation of such L-serine derivatives whereby synthesis of the L-erythro isomer of such serine derivatives may be selectively obtained. While the present invention is thus concerned with the preparation of L-serine derivatives, it also may be used to advantage in the aldol condensation of glycine with formaldehyde to produce L-serine. Accordingly as used herein, the term "L-serine derivative" includes L-serine itself as well as the various derivatives defined hereinafter in formula (I).
Serine hydroxymethyltransferase (alternatively referred to for the purposes of the subject application as "SHMT") is widely distributed in both eucaryotes and procaryotes and has been isolated from the livers of a variety of mammals and from various bacteria such as Escherichia coli and Clostridium cylindrosporum. Genetically engineered microorganisms which overproduce this enzyme in large quantities and thereby facilitate the preparation of pure enzyme have also been reported in the literature. See, Plamann et al., Nucleic Acids Res., Vol. 11, pages 2065-2075 (1983), Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985), and Hamilton et al., Trends in Biotechnology, Vol. 3, No. 1, pages 64-68 (1985).
SHMT from a variety of different sources has been reported to catalyze the reversible cleavable of betaphenylserines, including L-erythro-beta-phenylserine, to benzaldehyde (or substituted benzaldehyde) and glycine. See, Ulevitch et al., Biochemistry, Vol. 16, No. 24, pages 5342-5350 and 5356-5369 (1977); Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985); and Ching et al., Biochemistry, Vol. 18, No. 5, pages 821-829 (1979).
In Nakazewa et al., U.S. Pat. No. 3,871,958, there is disclosed a process for the preparation of L-serine derivatives of the formula: ##STR1## wherein R is an organic residue having at least two carbon atoms by reacting an aldehyde with glycine in aqueous solution at a pH of 5 to 10 and a temperature of 5 to 60.degree. C. in the presence of an enzyme obtained from microorganisms belonging to the genera Escherichia, Citrobacter, Klebsiella, Aerobacter, Serratio, Proteus, Bacillus, Staphylococcus, Arthrobacter, Bacterium, Xanthomonas, Candida, Debaryomyces, Corynebacterium and Brevibacterium. It is suggested that the active enzyme in this reaction is threonine aldolase. In order to improve yields, it is recommended that the amount of glycine in the reaction system be equimolar with or in excess of the aldehyde, and that the amount of aldehyde in the reaction system be limited to from 0.1 to 10% by weight of the reaction mixture.
A similar description of threonine aldolase for the preparation of L-beta-phenylserine is set forth in Japanese patent document SHO 54-3952, published Feb. 28, 1979.
A number of authors have also suggested that glycine may be condensed with formaldehyde to give L-serine using SHMT. See, Hamilton et al., Trends in Biotechnology, Vol. 3, No. 1, pages 64-68 (1985); and U.K. Published Patent Application No. 2130216A, filed Nov. 18, 1983).
European Patent Application No. 0 220 923, published May 6, 1987, corresponding to commonly assigned copending U.S. Patent application Ser. No. 789,595, filed Oct. 21, 1985, describes the use of SHMT obtained from a genetically engineered Escherichia coli strain transformed with the pGS29 plasmid for condensing benzaldehyde and glycine methyl ester to produce betaphenylserine methyl ester. The reaction conditions employed during the condensation reaction include a pH of from 6.5 to 9, a temperature of from 10 to 65.degree. C., a benzaldehyde concentration of from 10 to 100 mM and a glycine ester from 10 to 150 mM. While at a betaphenylserine methyl ester yield of 1.48 g/l, this process produced a beta-phenylserine methyl ester product containing as much as 83% erythro isomer, it has been found that as the yield is increased the amount of threo isomer present in the product increases until at commercial rates of production substantial amounts of threo isomer are present.
While the prior art has thus recognized that various enzymes can be employed to catalyze the aldol condensation of glycine and aldehydes to make L-serine derivatives, the processes of the prior art have suffered from a number of disadvantages. The use of excess glycine in the prior art processes has resulted in a L-serine derivative product containing large amounts of residual glycine. The presence of this glycine in the product not only leads to high raw material costs, but in addition, requires the use of a troublesome separation procedure in order to separate the glycine from the L-serine derivative, further adversely effecting the process economics.
Moreover, with the processes of the prior art, it has been found that the L-seine derivative formed comprises a mixture of optical isomers containing predominantly L-threo isomer at equilibrium. While for some purpose such mixtures are satisfactory, other purposes, such as the preparation of aspartame from L-phenylserine, require the use of only the L-erythro isomer of such L-serine derivatives. In end-uses of this latter type, the l-threo isomer is either non-reactive, or leads to contamination of desirable stereoisomers with undesirable stereoisomers.